1. Field of the Invention
The present invention relates generally to the RIN and MC genes. More specifically, it relates to methods and compositions for the modification of plant phenotypes with the RIN and MC genes.
2. Description of the Related Art
The ripe phenotype is the summation of biochemical and physiological changes occurring at the terminal stage of fruit development rendering the organ edible and desirable to seed dispersing animals and valuable as an agricultural commodity. These changes, although variable among species, generally include modification of cell wall ultrastructure and texture, conversion of starch to sugars, increased susceptibility to post-harvest pathogens, alterations in pigment biosynthesis/accumulation, and heightened levels of flavor and aromatic volatiles (Rhodes, 1980; Hobson and Grierson, 1993). Several of theses ripening attributes translate to decreased shelf-life and high input harvest, shipping and storage practices, particularly via changes in firmness and the overall decrease in resistance to microbial infection of ripe fruit. Currently acceptable techniques for minimizing the consequences of undesirable ripening characteristics include premature harvest, controlled atmosphere storage, pesticide application, and chemically induced ripening to synchronize the timing of maturation. Unfortunately, added production, shipping and processing expenses, in addition to reduced fruit quality, are often the consequence of these practices, challenging both the competitiveness and long term sustainability of current levels of crop production.
Although most fruit display modifications in color, texture, flavor, and pathogen susceptibility during maturation, two major classifications of ripening fruit, climacteric and non-climacteric, have been utilized to distinguish fruit on the basis of respiration and ethylene biosynthesis rates. Climacteric fruit such as tomato, cucurbits, avocado, banana, peaches, plums, and apples, are distinguished from non-climacteric fruits such as strawberry, grape and citrus, by their increased respiration and ethylene biosynthesis rates during ripening (Grierson, 1986). Ethylene has been shown to be necessary for the coordination and completion of ripening in climacteric fruit via analysis of inhibitors of ethylene biosynthesis and perception (Yang, 1985; Tucker and Brady, 1987), in transgenic plants blocked in ethylene biosynthesis (Klee et al., 1991; Oeller et al., 1991; Picton et al., 1993a), and through examination of the Never-ripe (Nr) ethylene perception mutant of tomato (Lanahan et al., 1994).
Considerable attention has been directed toward elucidating the molecular basis of ripening in the model system of tomato during recent years (reviewed in Spiers and Brady, 1991; Gray et al., 1992 and 1994; Giovannoni, 1993; Theologis 1992 and Theologis et al., 1993). The critical role of ethylene in coordinating climacteric ripening at the molecular level was first observed via analysis of ethylene inducible ripening-related gene expression (Tucker and Laties, 1984; Lincoln et al., 1987; Maunders et al., 1987; DellaPenna et al., 1989; Starrett and Laties; 1993). Several ripening genes, including ACC synthase and ACC oxidase, have been shown via antisense gene repression to have profound influences on the onset and degree of ripening (Hamilton et al., 1990; Oeller et al., 1991). Although the sum effect of this research has been a wealth of information pertaining to the regulation of ethylene biosynthesis and its role in ripening, the molecular basis of developmental cues which initiate ripening-related ethylene biosynthesis, and additional aspects of ripening not directly influenced by ethylene, remain largely unknown (Theologis et al., 1993).
Single locus mutations which attenuate or arrest the normal ripening process, and do not ripen in response to exogenous ethylene, have been identified in tomato and are likely to represent lesions in regulatory components necessary for initiation of the ripening cascade, including ethylene biosynthesis (Tigchelaar et al., 1978; Grierson, 1987; Giovannoni, 1993; Hobson and Grierson, 1993; Gray et al., 1994). One such mutation, the Nr mutation, has been identified and represents a gene responsible for ethylene perception and/or signal transduction and is a tomato homologue of the Arabidopsis Ethylene response I (EtrI) gene (Yen et al., 1995; Wilkinson et al., 1995).
Tomato has served as a model for ripening of climacteric fruit. Ripening-related genes have been isolated via differential gene expression patterns (Slater et al., 1985, Lincoln et al., 1987, Pear et al., 1989, Picton et al., 1993b) and biochemical function (DellaPenna et al., 1986; Sheehy et al., 1987; Ray et al., 1988; Biggs and Handa, 1989; Harriman and Handa, 1991; Oeller et al., 1991; Yelle et al., 1991). Promoter analysis of ripening genes has been performed via examination of promoter/reporter construct activities in transient assay systems and transgenic plants. The result has been the identification of cis-acting promoter elements which are responsible for both ethylene and non-ethylene regulated aspects of ripening (Deikiman et al., 1992; Montgomery et al., 1993). Trans-acting factors which interact with these promoters also have been identified via gel-shift and footprint experiments, although none have been isolated or cloned (Deikman and Fischer, 1988; Cordes et al., 1989; Montgomery et al., 1993).
The in vivo functions of several ripening-related genes including polygalacturonase, pectinmethylesterase, ACC synthase, ACC oxidase, and phytoene synthase have been tested via antisense gene repression and/or mutant complementation in transgenic tomatoes. For example, the cell wall pectinase, polygalacturonase, was shown to be necessary for ripening-related pectin depolymerization and pathogen susceptibility, however , the inhibition of PG expression had minimal effects on fruit softening (Smith et al., 1988, Giovannoni et al. 1989, Kramer et al., 1990). Significant reduction in rates of ethylene evolution resulting in inhibition of most ripening characteristics was observed in both ACC synthase and ACC oxidase antisense mutants (Oeller et al., 1991, Hamilton et al., 1990). Non-ripening antisense fruit were subsequently restored to normal ripening phenotype with the application of exogenous ethylene.
Further analysis of transgenic tomatoes inhibited in ethylene biosynthesis demonstrates that climacteric ripening represents a combination of both ethylene mediated and developmental control (Theologis et al., 1993). Although antisense ACC synthase tomatoes which failed to produce ethylene did not ripen, gene expression analysis demonstrated that several ripening-related genes, including polygalacturonase and E8 are expressed in the absence of ethylene. This observation confirms the presence of a developmental (or non-ethylene regulated) component of ripening. In fact, an ethylene requirement was observed for translation but not transcription of polygalacturonase mRNA, suggesting interaction between ethylene and non-ethylene components of ripening for expression of at least a subset of ripening genes (Theologis et al., 1993).
While the above studies have provided some insight into the ripening process in plants, there is still a great need in the art for novel methods and compositions for the creation of plants having enhanced phenotypes. In particular, there is a need in the art for the isolation the RIN and NOR genes. The isolation of these genes would allow the creation of novel transgenic plants altered in their fruit characteristics and/or ethylene responsiveness, and having one or more added beneficial properties.
In one aspect, the invention provides an isolated nucleic acid sequence comprising a RIN gene. In one embodiment of the invention, the RIN gene may be further defined as isolatable from the nucleic acid sequence of SEQ ID NO:6, from SEQ ID NO:5 or alternatively from SEQ ID NO:8, or any combinations of the foregoing.
In yet another aspect, the invention provides an isolated nucleic acid sequence comprising from about 17 to about 1172, about 25 to about 1172, about 30 to about 1172, about 40 to about 1172, about 60 to about 1172, about 100 to about 1172, about 200 to about 1172, about 400 to about 1172, about 600 to about 1172, about 800 to about 1172, or about 1000 to about 1172 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:8. In one embodiment of the invention, the isolated nucleic acid sequence has the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:8.
An isolated nucleic acid in accordance with the invention, for example, as described above and below, may further comprise an enhancer element. An exemplary enhancer comprises an intron. Such isolated nucleic acids may also comprise a native or heterologous transcriptional terminator or enhancers, as desired.
In still yet another aspect, the invention provides an expression vector comprising a RIN gene. The expression vector may comprise a native or heterologous promoter operably linked to the RIN gene in either sense or antisense orientation relative to said promoter. Any heterologous promoter may be used including a CaMV 35S, CaMV 19S, nos, Adh, actin, histone, ribulose bisphosphate carboxylase, R-allele, root cell promoter, xcex1-tubulin, ABA-inducible promoter, turgor-inducible promoter, rbcS, sucrose synthetase 1, alcohol dehydrogenase 1, pea small subunit RuBP carboxylase, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, bean glycine rich protein 1, CaMV 35s transcript, Potato patatin, actin, cab, PEPCase or S-E9 small subunit RuBP carboxylase promoter.
The expression vector may also comprise any selectable marker, for example, a selectable marker selected from the group consisting of phosphinothricin acetyltransferase, glyphosate resistant EPSPS, aminoglycoside phosphotransferase, hygromycin phosphotransferase, neomycin phosphotransferase, dalapon dehalogenase, bromoxynil resistant nitrilase, anthranilate synthase and glyphosate oxidoreductase. The expression vector may be circular or may comprise a linear nucleic acid segment, such as in the case of an expression cassette. In one embodiment of the invention, the expression vector is a plasmid vector. The expression vector may further include any other desired elements, such as an enhancer, a nucleic acid sequence encoding a transit peptide, or a heterologous terminator operably linked to said RIN gene, for example, a nos terminator. The RIN gene included in the expression vector may include any of the RIN-gene containing compositions described herein and exemplified in the claims. Such compositions may comprise any number of contiguous nucleotides from, for example, SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:8, as well as all possible combinations thereof The nucleic acid compositions also specifically include the open reading frames of the RIN cDNA sequences provided in SEQ ID NO:7, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:8.
In still yet another aspect, the invention provides a transgenic plant comprising a stably transformed RIN-gene containing expression vector in accordance with the invention. The plant may be any species of plant, for example, a tomato plant. In one embodiment of the invention, the transgenic plant is further defined as a fertile R0 transgenic plant. Also included in the invention is a seed of the fertile R0 transgenic plant, wherein said seed comprises said expression vector. In another embodiment of the invention, the transgenic plant is further defined as a progeny plant of any generation of a fertile R0 transgenic plant, wherein said R0 transgenic plant comprises said expression vector. Still further included in the invention is a seed of the progeny plant, wherein said seed comprises said expression vector.
In still yet another aspect, the invention provides a crossed fertile transgenic plant prepared according to the method comprising the steps of: (i) obtaining a fertile transgenic plant comprising a selected DNA comprising a RIN gene; (ii) crossing said fertile transgenic plant with itself or with a second plant lacking said selected DNA to prepare the seed of a crossed fertile transgenic plant, wherein said seed comprises said selected DNA, and (iii) planting said seed to obtain a crossed fertile transgenic plant. The invention also provides a seed of the crossed fertile transgenic plant, wherein said seed comprises said selected DNA. The plant may be of any species, including a tomato plant. The plant may also be inbred or hybrid.
In still yet another aspect, the invention provides a method of manipulating the phenotype of a plant comprising the steps of (i) obtaining an expression vector comprising a RIN gene in sense or antisense orientation; (ii) transforming a recipient plant cell with said expression vector, and (iii) regenerating a transgenic plant from said recipient plant cell, wherein the phenotype of said plant is altered based on the expression of said RIN gene in sense or antisense orientation. Any method of transformation may be used, including microprojectile bombardment, PEG mediated transformation of protoplasts, electroporation, silicon carbide fiber mediated transformation, or Agrobacterium-mediated transformation. In one embodiment of the invention, the step of transforming comprises Agrobacterium-mediated transformation and the plant is a tomato plant.
In still yet another aspect, the invention provides a method of plant breeding comprising the steps of (i) obtaining a transgenic plant comprising a selected DNA comprising a RIN gene; and (ii) crossing said transgenic plant with itself or a second plant. In one embodiment of the invention, said second plant is an inbred plant. The method may further comprise the steps of (iii) collecting seeds resulting from said crossing; (iv) growing said seeds to produce progeny plants; (v) identifying a progeny plant comprising said selected DNA; and (vi) crossing said progeny plant with itself or a third plant. In one embodiment of the invention, the second plant and said third plant are of the same genotype, and may be inbred plants.
In still yet another aspect, the invention provides a transgenic plant cell stably transformed with a selected DNA comprising RIN gene. The cell may be from any plant species including tomato. The selected DNA may comprise any of the RIN-gene compositions described herein, for example, an expression vector as described above.
In still yet another aspect, the invention provides a nucleic acid sequence comprising from about 17 to about 1138, about 25 to about 1138, about 30 to about 1138, about 40 to about 1138, about 60 to about 1138, about 100 to about 1138, about 200 to about 1138, about 400 to about 1138, about 600 to about 1138, about 800 to about 1138, or about 1000 to about 1138 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:3. In one embodiment of the invention, an isolated nucleic acid sequence is provided having the nucleic acid sequence of SEQ ID NO:3.
In one aspect, the invention provides an isolated nucleic acid sequence comprising a MC gene. In one embodiment of the invention, the MC gene may be further defined as isolatable from the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:7 or alternatively from SEQ ID NO:8, or any combinations of the foregoing.
In another aspect, the invention provides an isolated nucleic acid sequence comprising from about 17 to about 1049, about 25 to about 1049, about 30 to about 1049, about 40 to about 1049, about 60 to about 1049, about 100 to about 1049, about 200 to about 1049, about 400 to about 1049, about 600 to about 1049, about 800 to about 1049, or about 1000 to about 1049, contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:7 or SEQ ID NO:8. In one embodiment of the invention, the isolated nucleic acid sequence has the nucleic acid sequence of SEQ ID NO:7 or SEQ ID NO:8.
In yet another aspect, the invention provides an isolated nucleic acid sequence comprising from about 17 to about 1097, about 25 to about 1097, about 30 to about 1172, about 40 to about 1097, about 60 to about 1097, about 100 to about 1097, about 200 to about 1097, about 400 to about 1097 , about 6 00 to about 1097 , about 800 to about 1097 , or about 1000 to about 1097 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:2. In one embodiment of the invention, the isolated nucleic acid sequence has the nucleic acid sequence of SEQ ID NO:2.
An isolated nucleic acid in accordance with the invention, for example, as described above and below, may further comprise an enhancer element. An exemplary enhancer comprises an intron. Such isolated nucleic acids may also comprise a native or heterologous transcriptional terminator or enhancers, as desired.
In still yet another aspect, the invention provides an expression vector comprising a MC gene. The expression vector may comprise a native or heterologous promoter operably linked to the MC gene in either sense or antisense orientation relative to said promoter. Any heterologous promoter may be used including a MC, CaMV 35S, CaMV 19S, nos, Adh, actin, histone, ribulose bisphosphate carboxylase, R-allele, root cell promoter, xcex1-tubulin, ABA-inducible promoter, turgor-inducible promoter, rbcS, sucrose synthetase 1, alcohol dehydrogenase 1, pea small subunit RuBP carboxylase, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, bean glycine rich protein 1, CaMV 35s transcript, Potato patatin, actin, cab, PEPCase or S-E9 small subunit RuBP carboxylase promoter.
The expression vector may also comprise any selectable marker, for example, a selectable marker selected from the group consisting of phosphinothricin acetyltransferase, glyphosate resistant EPSPS, aminoglycoside phosphotransferase, hygromycin phosphotransferase, neomycin phosphotransferase, dalapon dehalogenase, bromoxynil resistant nitrilase, anthranilate synthase and glyphosate oxidoreductase. The expression vector may be circular or may comprise a linear nucleic acid segment, such as in the case of an expression cassette. In one embodiment of the invention, the expression vector is a plasmid vector. The expression vector may further include any other desired elements, such as an enhancer, a nucleic acid sequence encoding a transit peptide, or a heterologous terminator operably linked to said MC gene, for example, a nos terminator. The MC gene included in the expression vector may include any of the MC-gene containing compositions described herein and exemplified in the claims. Such compositions may comprise any number of contiguous nucleotides from, for example, SEQ ID NO:2, SEQ ID NO:7 and SEQ ID NO:8, as well as all possible combinations thereof. The nucleic acid compositions also specifically include the open reading frames of the MC cDNA sequences provided in SEQ ID NO:2, SEQ ID NO:7 and SEQ ID NO:8.
In still yet another aspect, the invention provides a transgenic plant comprising a stably transformed MC-gene containing expression vector in accordance with the invention. The plant may be any species of plant, for example, a tomato plant. In one embodiment of the invention, the transgenic plant is further defined as a fertile R0 transgenic plant. Also included in the invention is a seed of the fertile R0 transgenic plant, wherein said seed comprises said expression vector. In another embodiment of the invention, the transgenic plant is further defined as a progeny plant of any generation of a fertile R0 transgenic plant, wherein said R0 transgenic plant comprises said expression vector. Still further included in the invention is a seed of the progeny plant, wherein said seed comprises said expression vector.
In still yet another aspect, the invention provides a crossed fertile transgenic plant prepared according to the method comprising the steps of: (i) obtaining a fertile transgenic plant comprising a selected DNA comprising a MC gene; (ii) crossing said fertile transgenic plant with itself or with a second plant lacking said selected DNA to prepare the seed of a crossed fertile transgenic plant, wherein said seed comprises said selected DNA; and (iii) planting said seed to obtain a crossed fertile transgenic plant. The invention also provides a seed of the crossed fertile transgenic plant, wherein said seed comprises said selected DNA. The plant may be of any species, including a tomato plant. The plant may also be inbred or hybrid.
In still yet another aspect, the invention provides a method of manipulating the phenotype of a plant comprising the steps of: (i) obtaining an expression vector comprising a MC gene in sense or antisense orientation; (ii) transforming a recipient plant cell with said expression vector; and (iii) regenerating a transgenic plant from said recipient plant cell, wherein the phenotype of said plant is altered based on the expression of said MC gene in sense or antisense orientation. Any method of transformation may be used, including microprojectile bombardment, PEG mediated transformation of protoplasts, electroporation, silicon carbide fiber mediated transformation, or Agrobacterium-mediated transformation. In one embodiment of the invention, the step of transforming comprises Agrobacterium-mediated transformation and the plant is a tomato plant.
Still yet another aspect of the invention provides a method of plant breeding comprising the steps of (i) obtaining a transgenic plant comprising a selected DNA comprising a MC gene; and (ii) crossing said transgenic plant with itself or a second plant. In one embodiment of the invention, said second plant is an inbred plant. The method may further comprise the steps of: (iii) collecting seeds resulting from said crossing; (iv) growing said seeds to produce progeny plants; (v) identifying a progeny plant comprising said selected DNA; and (vi) crossing said progeny plant with itself or a third plant. In one embodiment of the invention, the second plant and said third plant are of the same genotype, and may be inbred plants.
Still yet another aspect of the invention provides a transgenic plant cell stably transformed with a selected DNA comprising MC gene. The cell may be from any plant species including tomato. The selected DNA may comprise any of the MC-gene compositions described herein, for example, an expression vector as described above.
In still yet another aspect, the invention provides an MC promoter. In one embodiment of the invention, the promoter is defined as being isolatable from the nucleic acid sequence of SEQ ID NO:8. In further embodiments of the invention, the MC promoter may comprise a nucleotide sequence defined by nucleotide 5695 to nucleotide 8237 of SEQ ID NO:8. Alternatively, in still further embodiments of the invention, the promoter may be defined as comprising a nucleotide sequence selected from the group consisting of nucleotide 5695 to nucleotide 6000 f SEQ ID NO:8, nucleotide 6001 to nucleotide 6400 of SEQ ID NO:8, nucleotide 6401 to nucleotide 6800 of SEQ ID NO:8, nucleotide 6801 to nucleotide 7200 of SEQ ID NO:8, nucleotide 7201 to nucleotide 7600 of SEQ ID NO:8, nucleotide 7601 to nucleotide 8000 of SEQ ID NO:8, or nucleotide 8001 to nucleotide 8237 of SEQ ID NO:8. The MC promoter may also be further defined as comprising a contiguous stretch of about 12, about 17, about 25, about 30, about 40, about 60, about 80, about 100, about 150, about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500 or about 2543 or longer segments of the nucleic acid sequence of SEQ ID NO:8, including all intermediate lengths therein. In one embodiment of the invention, the contiguous nucleic acids may be further defined as from a nucleotide sequence comprised on the nucleic acid sequence defined by nucleotide 5695 to nucleotide 8237 of SEQ ID NO:8. Alternatively, in particular embodiments of the invention, the nucleic acid segment may be from a nucleotide sequence selected from the group consisting of nucleotide 5695 to nucleotide 6000, nucleotide 6001 to nucleotide 6400 of SEQ ID NO:8, nucleotide 6401 to nucleotide 6800 of SEQ ID NO:8, nucleotide 6801 to nucleotide 7200 of SEQ ID NO:8, nucleotide 7201 to nucleotide 7600 of SEQ ID NO:8, nucleotide 7601 to nucleotide 8000 of SEQ ID NO:8, or nucleotide 8001 to nucleotide 8237 of SEQ ID NO:8.
In still yet another aspect, the invention provides MC promoter sequences included with additional elements, including an enhancer, a terminator, a selected coding region or any other desired sequences. Such sequences may be provided as an expression vector, or may comprise an expression cassette isolated from the vector. Further provided by the invention are plants transformed with nucleic acids comprising an MC promoter, as well as methods for making such plants.